The present invention relates to a method of isolating cells that have never before been identified and located from the mammalian central nervous system. The invention also relates to such isolated cells per se and to various uses thereof as well as assays using the same.
Until recent years, a xe2x80x98staticxe2x80x99 view on the fate of nerve cells in the central nervous system (CNS) was universally prevailing, based on the assumption that new neurons could not be generated in the adult mammalian brain. However, such renewal of neurons has been described in certain regions of the adult CNS, e.g. in the olfactory bulb, where signals from neurons from the organ of smell reach the brain (Kaplan et al., Science 197:1092) and in the dentate gyrus of hippocampus (Bayer et al., Science 216:890). Since neurons are unable to divide, the addition of new neurons suggested the existence of immature cells, i.e. progenitor or stem cells, which may generate neurons. Evidence supporting the existence of a multipotent neural stem cell in the adult mammalian CNS was presented a few years ago (Reynolds et al., Science 255:1707). However, as in several other organs, the realization of the existence of a stem cell has come before identifying and localizing the same. Interestingly, neurogenesis in the adult brain persists throughout adulthood in rodents (Kuhn P G, J. Neurosci. 16:20) and seems to be an evolutionary well conserved phenomenon present in a variety of mammals (Gould et al. J. Neurosci. 17:2492, Gould et al., Proc. Natl. Acad. Sci. USA 95:3168). In humans, the issue is difficult to address, although experimental data from cultures of adult human brain tissue (Kirschenbaum et al, Cereb. Cortex 5:576) suggest that there may be continuous neurogenesis also in the adult human CNS.
The existence of neural stem cells in the adult mammalian CNS was first demonstrated by culturing cells from the adult rat brain and spinal cord. Under certain culture conditions a population of multipotent neural stem cells can be propagated from dissociated adult rat brain and spinal cord (Reynolds et al., Science 255:1707, Dev. Biol. 175:1, Weiss et al., J. Neurosci. 16: 7599). The culture medium has to contain a mitogenic factor, e.g. epidermal growth factor (EGF) or fibroblast growth factor (FGF), and serum must be excluded. In contrast to stem cells, most other CNS cell types do not survive in these cultures.
Under these conditions, single cells proliferate in vitro and the progeny forms a cluster of aggregated cells (Reynolds et al., Science 255:1707, Dev. Biol. 175:1). Such cell clones detach from the culture dish after a few days in vitro. The cells continue to proliferate and form a characteristic spheroid cell aggregate, referred to as a neurosphere, of tightly clustered cells, all of which are derived from a single cell. Most of the cells in the neurosphere express nestin, an intermediate filament found in neuroepithelial stem cells. (Lendahl et al., Cell, 60:585), but not markers typical for differentiated cells. These undifferentiated cells rapidly differentiate if plated on an adhesive substrate or if serum is added to the culture medium. Importantly, a clone of cells derived from a single cell can generate neurons, astrocytes and oligodendrocytes, demonstrating that at least the initial cell was multipotent (Reynolds et al., Science 255:1707, ibid. Dev. Biol. 175:1). Moreover, if a cell clone is dissociated, many of the cells will form new clusters of undifferentiated multipotent cells (Reynolds et al., Dev. Biol. 175:1), thus fulfilling the criteria for being stem cells.
Thus, the method above suffers from the serious drawback that the cell population used is of a complex, mixed composition. Even though it has been possible to enhance the growth of some cell types, it is impossible to draw any conclusions regarding the original localization of the cells obtained.
Consequently, other methods have been proposed to determine the localization of the adult CNS stem cells, wherein different parts of the adult rodent forebrain have been carefully dissected and cultured to test for the capacity of neurogenesis. These studies have demonstrated that stem cells are most abundant in the wall of the lateral ventricle and in the hippocampus (Lois et al., Proc. Natl. Acad. Sci. USA, 90:2074, Morsehead et al., Neuron 13:1071, Palmer et al., Mol. Cell. Neurosci. 6:474, ibid, 8:389). Furthermore, stem cells can be isolated from the walls of the third and fourth ventricles as well as from the adult spinal cord, suggesting the presence of stem cells adjacent to the ventricular system along the entire neuraxis (Weiss et al., J. Neurosci. 16: 7599).
However, the exact localization and identity of the neural stem cell has been enigmatic. The wall of the lateral ventricles has been the subject of detailed morphological studies (Doetsch et al., J. Neurosci. 17:5046). The ventricular system is lined by a single layer of ependymal cells. Mammalian ependymal cells have traditionally been considered to be highly specialized cells with the main function to form a barrier between the nervous tissue and the cerebrospinal fluid (Del Bigio, Glia 14:1), which strongly argues against these cells being undifferentiated stem cells. Beneath the ependymal layer is the subependymal layer, also known as the subventricular zone. This area harbors astrocytes, neuroblasts and progenitor cells (Doetsch et al., J. Neurosci. 17:5046). The progenitor cells in the subependymal layer have a high proliferation rate (Morsehead et al., J. Neurosci. 12:249). Generally, stem cells proliferate very slowly and when the rapidly proliferating subependymal cells were selectively killed, the stem cell population was not depleted, arguing against these cells being the stem cells (Morsehead et al., Neuron 13:1071).
WO 97/44442 (Johe) discloses isolation of stem cells from the CNS of mammals and more specifically from the subependymal region of striatum lining the lateral ventricles. However, only subependymal cells are used and thus there is no further teaching regarding the identity and role of mammalian ependymal cells that alters the conventional one.
WO 95/13364 (Weiss, et al.) relates to a method of proliferation of CNS precursor cells located by the CNS ventricle of a mammal. However, only precursor cells are disclosed, and there are no teachings regarding other cell stages, such as stem cells.
In this context, it is interesting to note that besides the olfactory bulb and the hippocampus, data on continuous neurogenesis throughout adulthood in other regions of the mammalian brain have been scarce. As an example that neurogenesis may be a more widespread phenomenon, a small number of cells with the capacity to generate neurons in vitro has been isolated from the striatum and septum (Palmer et al., Mol. Cell. Neurosci. 6:474), although it has not been tested if these cells have stem cell properties or if they are committed neuronal progenitors.
There is increasing evidence that nervous system injuries may affect stem cells in the adult CNS. After both spinal cord and brain injuries, nestin expression is increased in cells lining the central canal and in the subventricular zone, respectively (Frisxc3xa9n et al., J. Cell Biol. 131:453, Holmin et al. Eur. J. Neurosci. 9:65). These cells have been suggested to derive from stem cells. With time, nestin expressing cells are seen progressively further from the central canal and the lateral ventricle and these cells express astrocytic markers (Frisxc3xa9n et al., J. Cell Biol. 131:453, Holmin et al. Eur. J. Neurosci. 9:65). These data have lead to the suggestion that stem cells or progenitor cells residing by the ventricular system are induced to proliferate, migrate toward the site of the injury and differentiate to astrocytes. Furthermore, hippocampal lesions increase the proliferation of hippocampal progenitor cells and the number of granular neurons in the hippocampus (Gould et al. Neurosci. 80:427). However, since the stem cell has not been identified or exactly localized it is not clear whether stem cells play a role in injury processes.
Cell loss is a common factor in many types of nervous system disorders. Distinct cell types are affected in different diseases, e.g. dopaminergic neurons in Parkinson""s disease, motor neurons in amyotrophic lateral sclerosis and oligodendrocytes in multiple sclerosis. Several different cell types in a certain area can be affected in other situations, such as stroke or traumatic injury. Currently, no methods are available in clinical practice to stimulate generation of new cells in the nervous system. Transplantation of cells from human embryos or animals have been tested clinically with some encouraging results. However, these methods have several problems, mainly ethical and immunological, which makes it very unlikely that they will be used in any larger number of patients.
Accordingly, the discovery of the existence of neural stem cells in the adult CNS of mammals is important and may make it possible to develop strategies to stimulate generation of new neurons or glial cells. However, several important questions have remained unanswered and better methods to culture these cells and to study them quantitatively in vivo are needed. Most importantly, it is absolutely vital to identify and localize the stem cell in the adult CNS in order to be able to study these cells further and to stimulate generation of new neurons from the stem cells.
Furthermore, there are no methods available today to purify stem cells at an early step in tissue culture. Although there are several general methods available for purifying cell populations in other tissues, it is impossible to utilize these methods, or to develop new methods, without knowledge of the true identity of the stem cell. Such methods would allow studies of a more well defined cell population and would be valuable for screening pharmaceutical compounds. Moreover, the development of quantitative methods to label and follow the stem cells and their progeny in vivo to allow detailed studies of, for example, regulation of the generation of new neurons to analyze the effect of different chemicals or genetically manipulate the stem cells are needed. Again, although there are methods known in the art that can be used to follow other cell populations in vivo, it is impossible to utilize these methods or develop new methods for following stem cells since the identity of the stem cell has been unknown. The development of quantitative methods to follow stem cells and their progeny in animal models of neurodegenerative disorders and injuries of the CNS would open up the possibility to screen new treatment strategies in human conditions where today only some of the symptoms, but not the neuronal loss per se, can be alleviated.
Thus, a problem within this field is that even though neural stem cells are known to exist, the localization and identity therof is not known. Could this be accomplished, a great step forward would be taken by research aimed at providing the above defined goals.
The object of the present invention is solve the above defined problem. More specifically, the present invention relates to a method of isolating ependymal neural stem cells from a mammalian CNS. The present identification of the stem cell has now made it possible to develop methods to purify these cells, study them quantitatively in vivo, genetically modify them and stimulate them with various pharmaceutical compounds in vitro and in vivo. Furthermore, the present invention provides evidence that the stem cells follow new migratory pathways to various neuroanatomical cell groups in the CNS and that they are able to transform into neurons in vivo. The invention also comprises an unbiased quantitative method to assess neurogenesis in various regions of the brain as well as techniques to analyze the total number of stem cells and their progeny migrating to various regions of the brain. Altogether, the developments provided by the present invention greatly increase the possibilities to develop strategies to stimulate generation of new cells in the central nervous system.
Accordingly, the actual identity and localization of ependymal neural stem cells are disclosed herein for the first time ever, thus enabling various advantageous uses and applications thereof within the medical and diagnostic field. The invention also relates to in vitro and in vivo assays, wherein the new findings according to the invention are advantageously employed.